Production and extraction of procyanidins from plant cell cultures

Abstract

Provided herein are methods of making cocoa polyphenol preparations, which methods comprises harvesting cocoa polyphenols, for example, procyanidins, from the cell suspension culture. In examples of these methods, the resultant cocoa polyphenol preparation is substantially (or in some cases, completely) free of detectable caffeine and theobromine, and more generally substantially free of xanthine alkaloids. Methods of producing a cell suspension culture of cacao cells are also described, including cell suspension cultures useful for making cocoa polyphenol and, more specifically, procyanidin preparations. Theobroma and Herrania sp cell suspension cultures and cocoa polyphenol preparations made therefrom are also provided, in particular xanthine alkaloid-free (or caffeine- and/or theobromine-free) cocoa polyphenol preparations.

We claim: 
     
       1. A method of producing a cell suspension culture of cacao cells, comprising:
 growing callus from a  Theobroma cacao  plant on growth medium to form a callus culture; 
 identifying and selecting a rapidly growing cell line from the cells of the  Theobroma cacao  callus culture, the rapidly growing cell line selected to produce procyanidins in cell suspension with less than 2% theobromine and/or less than 0.5% caffeine contamination on a dry weight basis; and 
 initiating the cell suspension culture by inoculating the rapidly growing cell line into liquid medium and producing in cell suspension procyanidins with less than 2% theobromine and/or less than 0.5% caffeine contamination on a dry weight basis. 
 
     
     
       2. The method of  claim 1 , wherein the  Theobroma cacao  plant is a floral explant selected from the group staminode, sepal and petal base explants. 
     
     
       3. The method of  claim 1 , further comprising growing the cell suspension culture in flasks, any suitable culture vessels, or bioreactors. 
     
     
       4. The method of  claim 3 , wherein the growing is effected in vessels or bioreactors and in batch, fed-batch or continuous mode. 
     
     
       5. A  Theobroma cacao  cell suspension culture produced by the method of  claim 1 . 
     
     
       6. The method of  claim 1 , wherein the growth medium is the liquid media XXXIII listed in Table 1. 
     
     
       7. The method of  claim 1 , wherein the callus is from a vegetative material. 
     
     
       8. The method of  claim 7 , wherein the vegetative material is selected from leaves, stems, meristem, nodes, or internodes that are young or mature. 
     
     
       9. The method of  claim 1 , wherein the step of selecting the rapidly growing cell line includes analyzing the callus culture using high performance liquid chromatography to determine the production of procyanidins.